Method for capturing analytes eluted from surface-bound ligands

ABSTRACT

Methods for capturing analytes associated with surface-bound ligands are disclosed. The methods involve eluting analytes from surface-bound ligands with a first liquid to generate free analytes, and capturing the free analytes with a solid capturing material within the first liquid to generate a first liquid containing captured analytes. The first liquid may be a flowing liquid or a non-flowing liquid, and the surface to which the surface-bound ligand is attached may be a sensing surface, such as a biosensor, or a non-sensing surface. The captured analytes may be further consolidated at a location removed from the surface-bound ligand, eluted from the solid capturing material with a second liquid, and used for subsequent analysis or procedures.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 60/190,336 filed Mar. 16, 2000, which provisionalapplication is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates generally to methods for capturing analytesassociated with surface-bound ligands and, more specifically, to use ofa solid capturing material for capturing analytes eluted fromsurface-bound ligands.

BACKGROUND OF THE INVENTION

A variety of analytical techniques are used to characterize interactionsbetween molecules, particularly in the context of assays directed to thedetection of biomolecular interactions. For example, antibody:antigeninteractions are of fundamental importance in many fields, includingbiology, immunology and pharmacology. In this context, many analyticaltechniques involve binding of a “ligand” (such as an antibody) to asolid support, followed by contacting the ligand with an “analyte” (suchas an antigen). Following contact of the ligand and analyte, somecharacteristic is measured which is indicative of the interaction, suchas the ability of the ligand to bind the analyte. After measurement ofthe interaction, the ligand:analyte pair is typically disrupted with anelution and/or regeneration solution in order to regeneratesurface-bound ligand for further analytical measurement.

The freed analyte of the ligand:analyte pair, however, is commonly notreused; rather, the freed analyte is typically disposed of together withthe elution and/or regeneration solution. This practice is undesirablebecause researchers very often have only limited quantities of theanalyte for analytical measurement purposes, and because researchersvery often desire to perform further analytical measurements directed tothe analyte itself. Accordingly, there is a need in the art toeffectively consolidate freed analyte from a ligand:analyte pair suchthat the freed analyte is amenable to subsequent analytical measurement.

The need to effectively consolidate freed analyte for subsequentanalytical measurement may be illustrated in the context of biosensorswhich use surface plasmon resonance (SPR) to monitor the interactionsbetween an analyte and a ligand bound to a solid support. In thisregard, a representative class of biosensor instrumentation is sold byBiacore AB (Uppsala, Sweden) under the trade name BIAcore® (hereinafterreferred to as “the BIAcore instrument”). The BIAcore instrumentincludes a light emitting diode, a sensor chip covered with a thin goldfilm, an integrated microfluidic cartridge and photo detector. Incominglight from the diode is reflected in the gold film and detected by thephoto detector. At a certain angle of incidence (“the SPR angle”), asurface plasmon resonance wave is set up in the gold layer, which isdetected as an intensity loss or “dip” in the reflected light. Thetheoretical basis behind the BIAcore instrument has been fully describedin the literature (see, e.g., Jönsson, U. et al., Biotechniques11:620-627 (1991)).

In addition to SPR analysis using the BIAcore instrument, researchersare beginning to appreciate the synergistic effects of coupling SPRtechnology with other analytical techniques. In this context, thereal-time interaction analysis offered by the BIAcore instrumentcomplements other known methods for investigating both biomolecularstructure and function. For example, SPR has recently been coupled withmass spectroscopy (i.e., SPR-MS) to provide an extremely powerfulmicropreparative technique for biomolecular investigations (see, e.g,PCT International Publication No. WO 97/09608). In connection withSPR-MS, analyte is freed from the surface-bound ligand bymatrix-assisted laser desorption/ionization for subsequent analyticalmeasurement by mass spectrometry.

One of the problems posed by eluting analyte away from surface-boundligands for subsequent analytical measurements is that substantialamounts of analyte can be lost due to nonspecific binding of analyte tothe walls and other components of the microfluidic cartridge as theelution and/or regeneration solution flows through the microfluidiccartridge. Moreover, once eluted away from surface-bound ligands,analyte must still be consolidated so that there will be enough samplefor subsequent analysis. Accordingly, there is a need in the art forimproved methods and micropreparative techniques for consolidatingbiomolecules associated with surface-bound ligands. The presentinvention fulfills these needs, and provides further related advantages.

SUMMARY OF THE INVENTION

In brief, the present invention is directed to methods for capturing ananalyte associated with a surface-bound ligand, as well as to methodsfor consolidating the same. In one embodiment, the method involveseluting the analyte from the surface-bound ligand by contacting thesurface-bound ligand with a first liquid flow that dissociates theanalyte from the surface-bound ligand to generate a free analyte withinthe first liquid flow. The free analyte is then captured by a solidcapturing material that is carried within the first liquid flow,yielding a first liquid flow containing captured analyte. The surface towhich the surface-bound ligand is attached may be either a sensingsurface, such as a sensing surface of a biosensor, or a non-sensingsurface.

In an alternative embodiment, the method involves eluting the analytefrom the surface-bound ligand on a surface of a biosensor by contactingthe surface-bound ligand with a first liquid that dissociates theanalyte from the surface-bound ligand to generate a free analyte withinthe first liquid. The free analyte is then captured by a solid capturingmaterial that is within the first liquid, yielding a first liquidcontaining captured analyte. In this embodiment, the surface to whichthe surface-bound ligand is attached is a surface of a biosensor, andthe first liquid may be a flowing or non-flowing liquid.

In both of the above embodiments, the captured analyte may be furtherconsolidated with similarly captured analytes at a location removed fromthe surface-bound ligand. Such consolidation may, for example, beaccomplished by passing the captured analytes of the first liquidthrough a separation device that prevents passage of the capturedanalytes, but allows passage of the first liquid. Once consolidated, thecaptured analytes may be contacted with a second liquid that elutes theanalyte of the captured analyte from the solid capturing material toyield free analyte, which may then be used in subsequent analyticaltechniques or procedures.

These and other aspects of the present invention will be evident uponreference to the following detailed description. To this end, variousreferences are cited throughout this application to further illustratespecific aspects of this invention. Such documents are each incorporatedherein by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention is directed to methods forcapturing an analyte associated with surface-bound ligand with a solidcapturing material. In a first embodiment, the solid capturing materialis carried within a first liquid flow and the surface to which thesurface-bound ligand is attached is a sensing or non-sensing surface. Ina second embodiment, the solid capturing material is within a firstliquid (flowing or non-flowing) and the surface to which thesurface-bound ligand is attached is the surface of a biosensor.

In the first embodiment, a method is disclosed for capturing an analyteassociated with a surface-bound ligand by eluting the analyte from thesurface-bound ligand by contacting the surface-bound ligand with a firstliquid flow that dissociates the analyte to generate a free analytewithin the first liquid flow. For example, a surface that has beenutilized for capturing a solubilized biomolecule (e.g., “real-time”monitoring of analyte-ligand biomolecular interactions with a biosensor)will have an analyte associated with its surface-bound ligand. Theanalyte is typically associated (e.g., bound) to the ligand bynon-covalent forces (such as electrostatic and Lewis acid-Lewis baseforces). In the context of this invention, the agent bound to thesurface is referred to as a “surface-bound ligand”, while the agent thatassociates with the surface-bound ligand is referred to as an “analyte.”

To this end, the terms “ligand” and “analyte” are to be construedbroadly, and encompass a wide variety of molecules ranging from smallmolecules to large proteins, as well as a variety of interaction pairs.For example, representative analyte:ligand interaction pairs include,but are not limited to, the following (wherein the analyte is listedfirst, followed by the ligand with which the analyte is associated, thenames of the analyte and ligand being separated by a colon):antigen:antigen-specific antibody, antigen-specific antibody:antigen,hormone: hormone receptor, hormone receptor:hormone,polynucleotide:complementary polynucleotide, avidin/streptavidin:biotin,biotin:avidin/streptavidin, enzyme:enzyme substrate or inhibitor, enzymesubstrate or inhibitor:enzyme, lectins:specific carboxyhydrate, specificcarboxyhydrate:lectins, lipids:lipid binding proteins ormembrane-associated proteins, lipid binding proteins ormembrane-associated proteins:lipids, polynucleotides:polynucleotidebinding proteins, polynucleotide binding proteins:polynucleotides,receptor:transmitter, transmitter:receptor, drug:target, target:drug, aswell as more general types of interactions such as protein:protein,protein:polynucleotide, polynucleotide:protein, DNA:DNA, DNA:RNA, andRNA:DNA interactions. Moreover, it is to be understood that the analytemay come from a single source, a mixture of natural compounds, a genelibrary, a mRNA or protein displayed gene library, or a chemical libraryof any kind.

Thus, in accordance with the practice of this invention, the analyte iseluted from the surface-bound ligand by contacting the same with a firstliquid flow that dissociates the analyte from the surface-bound ligand.Such elution or dissociation of the analyte from the surface-boundligand may be accomplished by use of any number of suitable elutionliquids or regeneration solutions (referred to herein as the “firstliquid” and as the “second liquid”). For example, aqueous solutionscomprising at least one acidic, basic, ionic, organic, detergent orchelating agent may be utilized as the first and second liquid. Suchaqueous solutions include those described in the journal articleentitled Identification and Optimization of Regeneration Conditions forAffinity-Based Biosensor Assays (Andersson, K et al., Anal. Chem.71(13):2475-81 (Jul. 1, 1999)), which article is incorporated herein byreference in its entirety.

More generally, and has been reported in the literature, various classesof analyte:ligand systems may be disrupted under the following exemplaryconditions: (1) Antibody:antigen interaction pairs—to varying degreeswith hydrochloric acid (HCl) of different concentrations (Malmborg etal, Scandinavial Journal of Immunology 35:643-50, 1992; Ward et al.,Biochemistry International 26:559-65, 1992) or with weaker acids,typically phosphoric or formic (Corr et al., Journal of ExperimentalMedicine 178:1877-92, 1993; VanCott et al., Journal of ImmunologicalMethods 183:103-17, 1995), or with detergent or chaotropic solutions(Tanchou et al., AIDS Research and Human Retroviruses, 10:983-93 1994;End et al., Journal of Biological Chemistry 268:10066-75, 1993); (2)Receptor:transmitter interaction pairs—with acids (Morelock et al.,Journal of Medicinal Chemistry 38:1309-18, 1995), bases (Lemmon et al.,Journal of Biological Chemistry 269:31653-58, 1994), under chaotropicconditions and high ion strength (Stitt et al., Cell 80:661-70, 1995),or under natural dissociation conditions (Ma et al., Journal ofBiological Chemistry 39:24430-36, 1994); (3) DNA interaction pairs—undervery mild regeneration conditions using detergents, EDTA, or undernatural dissociation conditions (Cheskis et al., Molecular Endocrinology1996; Casasnovas Journal of Biological Chemistry 270:13216-24, 1995);and (4) Glycoprotein interaction pairs—under acid conditions or usingsugar solutions (Okazaki et al., Journal of Molecular Recognition8:95-99 1995). The precise conditions for eluting the analyte from thesurface-bound ligand will, of course, depend upon the system underinvestigation. However, such conditions may readily be determined bythose having skill in the art.

In the first embodiment of the present invention, the first liquid is inflowing contact with surface-bound ligand (referred to herein as the“first liquid flow”). Suitable devices for contacting the first liquidflow with the surface-bound analyte are known in the art, and aregenerally referred to as fluidic delivery systems. A representativefluidic delivery system is the integrated microfluidic cartridgeutilized in the BIAcore instrument discussed previously in theBackground section, and which is capable of precisely and controllablyflowing a liquid over a surface. Such delivery systems are known in theart, such as described by U.S. Pat. Nos. 5,313,264 and 5,443,890 (bothof which are incorporated herein by reference).

Carried within the first liquid flow is a solid capturing material whichis capable of capturing the free analyte eluted from the surface-boundligand. In other words, the solid capturing material is allowed to flowtogether with the first liquid within the fluidic flow channel(s) thatbring the first liquid flow into contact with the surface-bound ligand.In this manner, the solid capturing material captures, typically byadsorption or absorption, the analyte in close proximity to thesurface-bound ligand from which it is eluted, thereby reducing theamount of free analyte lost due to nonspecific binding, such as bindingof free analyte to the walls and other components of the fluidicchannel(s). Because of the small size typically associated with fluidicflow channels, exemplary solid capturing material typically constitutesseparation beads of a small diameter (e.g., 2 to 10 micrometers).Further, the solid capturing material is generally selected to besuitable with the analyte:ligand system under investigation—that is, thematerial should be of a type that will readily capture the elutedanalyte. In this regard, there are a wide variety of solid capturematerials that meet these parameters.

As mentioned above, exemplary solid capturing materials are separationbeads, such as chromatographic beads used in liquid column adsorptionchromatography, especially chromatographic beads used in highperformance liquid chromatography (HPLC). However, the present inventionis not limited to chromatographic beads. Rather, any solid capturingmaterial adapted to the separation of solute in a solution on the basisof physicochemical properties may be employed. Accordingly, the solidcapturing materials of the present invention are inclusive of allchromatographic media, as well as other solid or semi-solid supportshaving similar properties (such as polymer-based particulate solidsupports). Therefore, the term “solid capturing material,” as usedwithin the context of the present invention, is to be construed broadlyand is inclusive of essentially any solid or semi-solid support made ofa synthetic, semi-synthetic and/or naturally occurring organic polymer,wherein such polymer(s) has the ability to adsorb analytes that havebeen freed from surface bound ligands; it also encompasses variousinorganic materials having like properties. Preferably, however, thesolid capturing materials of the present invention are spherical inshape, comprise a silica gel material having an amorphous structure, andare somewhat porous. The solid capturing material may also be magneticin nature, such as magnetic beads (especially useful for subsequentelution via ionization as in MALDI). Moreover, the solid capturingmaterials of the present invention may be derivatized with a wide rangeof chemical functionalities, as is appreciated by those skilled in theart, for specifically adsorbing the freed analyte of interest. Exemplaryin this regard are bead materials made from agarose, dextran,hydroxyapatit, silica, polyacrylamid, and hydrophilic polymers incross-linked form, which bead materials may also be porous, nonporous,and/or dense.

Thus, in one aspect of the present invention, the solid capturingmaterial are separation beads carried within the first liquid flow. Inone embodiment, a plurality of separation beads carried within the firstliquid flow are pumped through the one or more flow cells of abiosensor, such as the flow cells of the BIAcore instrument. Preferably,the flow rate is such that the first liquid flow is larninar. As isappreciated by those skilled in the art, a laminar flow rate will tendto centrally concentrate the separation beads carried within the flowliquid stream (i.e., the beads will generally tend to flow in the centerof the channel). This phenomenon (also known as hydrodynamic focusing)is due to the shear forces exerted by the wall of conduit onto theflowing liquid. The shear forces will cause a flow rate gradient acrossthe flow channel; a flowing bead will tend to flow centrally so as tohave the same or symmetrical flow forces on both of its sides.

In the first embodiment of this invention, the surface to which thesurface-bound ligand is attached may be either a sensing surface or anon-sensing surface. Thus, a sensing surface in accordance with thepresent invention may be a sensing surface of a biosensor; such asensing surface comprises a solid metal support (e.g., gold or silver)having a coating of a densely packed organic monolayer thereon (as isdisclosed in U.S. Pat. No. 5,436,161, which is incorporated herein byreference in its entirety.) The sensing surface may further comprise abiocompatible porous matrix like, for example, a hydrogel (e.g., apolysaccharide such as dextran) coupled to the organic monolayer coatingso to be operable in association with a biosensor.

As is appreciated by those skilled in the art, a biosensor is ananalytical device for analyzing minute quantities of sample solutionhaving an analyte of interest, wherein the analyte is analyzed by adetection device that may employ any one of a variety of detectionmethods. Typically, such methods include, but are not limited to, massdetection methods, such as piezoelectric, optical, thermo-optical andsurface acoustic wave (SAW) device methods, and electrochemical methods,such as potentiometric, conductometric, amperometric and capacitancemethods. With regard to optical detection methods, representativemethods include those that detect mass surface concentration, such asreflection-optical methods, including both internal and externalreflection methods, angle, wavelength or phase resolved, for exampleellipsometry and evanescent wave spectroscopy (EWS), the latterincluding surface plasmon resonance (SPR) spectroscopy, Brewster anglerefractometry, critical angle refractometry, frustrated total reflection(FTR), evanescent wave ellipsometry, scattered total internal reflection(STIR), optical wave guide sensors, evanescent wave-based imaging, suchas critical angle resolved imaging, Brewster angle resolved imaging, SPRangle resolved imaging, and the like. Further, photometric methods basedon, for example, evanescent fluorescence (TIRF) and phosphorescence mayalso be employed, as well as waveguide interferometers. While certainaspects of the present invention are hereinafter illustrated in thecontext of the BIAcore instrument (Biacore AB, Uppsala, Sweden) with itsSPR-based technology, it is to be understood that the present inventionis not limited to such systems.

In a second embodiment of this invention, a method is disclosed forcapturing an analyte associated with a surface-bound ligand on a surfaceof a biosensor by eluting the analyte from the surface-bound ligand togenerate a free analyte within a first liquid, and capturing the freeanalyte with a solid capturing material within the first liquid togenerate a first liquid containing captured analyte. This embodiment ispracticed in the same manner as disclosed above, except that the firstliquid may be either a flowing or non-flowing liquid when in contactwith the surface-bound ligand. To the extent that the first liquid is aflowing liquid, this embodiment represents a specific aspect of thefirst embodiment wherein the surface to which the surface-bound ligandis attached is a biosensor surface, and is fully described above. Incontrast, when the first liquid is a non-flowing liquid, the firstliquid may be contacted with the surface-bound ligand by any number ofprocedures and/or devices, including, for example, stop-flow fluidicliquid delivery techniques, as well as simple aspiration of the firstliquid onto (and off of) the surface-bound ligand of the biosensor. Inthis embodiment, the solid capturing material is within the first liquidat the point of capturing the free analyte, but need not have beencarried within the first liquid. Rather, the solid capturing materialmay, for example, be added to the first liquid after the step ofeluting.

Accordingly, in this aspect of the invention, it should be understoodthat the term “biosensor” covers not only analytical devices that useflow systems to contact the first liquid with the surface-bound ligand,such as the integrated microfluidic cartridge of the BIAcore instrument,but also includes analytical devices that use non-flow systems tocontact the first liquid with a sensing surface, such as a sensingsurface on the bottom of a cuvette. As such, this aspect of the presentinvention is applicable to both flow and non-flow biosensor systems.

In both the first and second embodiments of this invention, andfollowing capture of the free analyte by the solid capture material togenerate the first liquid containing captured analyte and more typicallya plurality of captured analytes, the captured analytes are consolidatedat a location remote from the surface-bound ligand. Such consolidationmay, for example, be accomplished by utilizing a column (such as acolumn used in micropreparative HPLC) that traps the solid capturingmaterial (e.g., the separation beads), but allows passage of the firstliquid. For example, in the embodiment wherein the first liquid is inflowing contact with the surface-bound ligand, a column may beoperatively connected to an exit portal so as to receive the flowingfirst liquid following contact with the surface-bound ligand. The columnmay be sieved in a manner so as to trap the solid separation beads—butallow passage of the first liquid. In this manner, the separation beadshaving captured analyte will consolidate within the column, whereas thefirst liquid will be discharged.

The consolidation step, however, is not limited to columns; rather, anytechnique that at least partially separates the solid capturing materialfrom the first liquid may be employed in the practice of the presentinvention. Exemplary in this regard is any other device capable ofaggregating the solid capturing material, such as decanting devices thatallow the solid capturing material to settle, centrifuge devices thatallow the first liquid to be separated from the solid capturingmaterial, and filtering devices that prevent passage of the solidcapturing materials but allow passage of the first liquid, and devicesfor attracting magnetic beads.

In still another aspect, and following consolidation within the columnor other consolidation device, the captured analyte may be contactedwith a second liquid so as to elute the bound analyte. Thus, and in oneembodiment, a second liquid is allowed to pass through the column havingthe plurality of captured analytes therein to thereby free the analyte.Because the solid capturing material is consolidated within the column,the freed analyte in the second eluent is typically of suchconcentration so as to be useful for a further analytical measurement,such as mass-spectroscopy, as well as for other uses (e.g., otheranalytical techniques or procedures). As with selection of the firstliquid, the selection of the second liquid depends upon the system underinvestigation (i.e., depends upon the nature of the coupling forcebetween the analyte and the solid capturing material). As discussedabove in the context of disrupting analyte:ligand pairs, proper elutionconditions for various analyte-solid capturing material combinations mayreadily be determined by those having skill in the art.

In still yet another aspect, and following consolidation within thecolumn or other consolidation device, the captured analyte may besubjected to matrix-assisted laser desorption/ionization time-of-flightmass spectrometry. In this embodiment, the solid capturing material mayhave an outer surface that comprises an appropriate matrix (e.g.,nicotonic or sinapinic acid). In general, an analyte captured on or insuch a matrix is amenable to intact desorption by laser irradiation asis appreciated by those skilled in the art.

The following examples are offered by way of illustration, and notrestriction.

EXAMPLES Example 1

Biosensor instrument: BIACORE® 3000 (Biacore AB, Uppsala, Sweden)

Sensor chip: CM 5 (Biacore AB, Uppsala, Sweden)

Coupling reagent: Amine Coupling Kit (Biacore AB, Uppsala, Sweden)

Capturing molecule: HIV protease Q7K 981126 (provided by UppsalaUniversity; Dr. Helena Danielsson)

Analyte: HIV inhibitor Saquvinavir (provided by Medivir AB, Sweden)

Capturing media: RP 2 Porous 10 micrometer diameter reversed phase media(PerSeptive Biosystems, CA, U.S.A.)

Elution liquid: 2% Formic acid (prepared from 98-100% formic acid; PARiedel de Haen, Germany)

Mass spectrometer: Bruker Biflex III™ (Bruker Daltonics Inc., U.S.A.)

The capturing molecule is immobilized to the sensor chip using theinstrument supplier's standard protocol for amine coupling resulting inca 4000 RU per flowcell relative to the baseline in a serial injectionsof flowcells 4, 3, 2 in BIACORE 3000. Analyte is then injected, theimmobilized amount of HIV protease being able to capture 30-100 RU ofHIV inhibitor—corresponding to 50-150 femtomole inhibitor per flowcell.

The captured analyte is recovered by using 4 μl slurry of a 2-4% slurryof chromatographic beads in 2% formic acid which is injected using the“MICRORECOVER” command in BIACORE 3000. The beads are equilibrated in agelloader tip according to Gobom et al., J. Mass. Spectrom. 34:105-116,1999, and then dispensed in about 50 μl 2% formic acid prior toMICRORECOVER. Recovered beads with the captured analyte are thentransferred manually with an Eppendorff pipette on top of a gelloadertip with already equilibrated RPC gel according to Gobom et al., supra.A washing step involving elution with 5% acetonitrile (ACN) in 0.1%trifluoroacetic acid (TFA) is then performed, and the analyte issubsequently eluted with 45% ACN/0.1% TFA saturated with α-cyanocinnamicacid directly onto a MALDI probe tip according to Gobom et al., supra.The presence of recovered analyte is then verified by MALDI MS performedon the Bruker BiflexIII mass spectrometer in reflectron mode.

Example 2

The procedure described in Example 1 above is followed, except that ascapturing media are used Micromer®-M C8, C18 reversed phase, 8 μmdiameter magnetic beads (Micromod Partikeltechnologie GmbH, Rostock,Germany). Prior to injection into the BIACORE 3000 by the MICRORECOVERcommand, the chromatographic magnetic beads are equilibrated in 2%formic acid by filtration using a batch procedure. A 2-4% slurry of theequlibrated beads are then prepared in 2% formic acid and transferred toan autosampler vial for injection into the BIACORE 3000.

While the present invention has been described in the context of theembodiments illustrated and described herein, the invention may beembodied in other specific ways or in other specific forms withoutdeparting from its spirit or essential characteristics. Therefore, thedescribed embodiments are to be considered in all respects asillustrative and not restrictive. The scope of the invention is,therefore, indicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

What is claimed is:
 1. A method for capturing an analyte, comprising thesequential steps of: contacting the analyte with a biosensor surface,the surface having a ligand bound thereto, so as to form an ananalyte:surface-bound ligand interaction pair; detecting the binding ofthe analyte to the surface-bound ligand; eluting the analyte from thesurface-bound ligand by contacting the biosensor surface with a firstliquid that dissociates the analyte from the surface-bound ligand togenerate a free analyte within the fist liquid; capturing the freeanalyte with a particulate solid capturing material within the firstliquid, to thereby generate a first liquid containing captured analyte;removing the particulate solid capturing material, having the capturedanalyte associated therewith, to a location remote from the biosensorsurface and consolidating the particulate solid capturing material,having the captured analyte associated therewith, at said location; andfreeing the analyte from the particulate solid capturing material. 2.The method of claim 1 wherein the analyte:surface-bound ligandinteraction pair is selected from the group consisting ofantigen:antigen-specific antibody, antigen-specific antibody:antigen,hormone:hormone receptor, hormone receptor:hormone,polynucleotide:complementary polynucleotide, avidin/streptavidin:biotin,biotin:avidin/streptavidin, enzyme:enzyme substrate, enzyme:enzymeinhibitor, enzyme substrate:enzyme, enzyme inhibitor:enzyme,lectins:carboxyhydrate, carboxyhydrate:lectins, lipid:lipid bindingprotein, lipid:membrane-associated protein, lipid binding protein:lipid,membrane-associated protein:lipids, polynucleotide:polynucleotidebinding protein, polynucleotide binding protein:polynucleotide,receptor:transmitter, transmitter:receptor, drug:target, target:drug,protein:protein, protein:polynucleotide, polynucleotide:protein,DNA:DNA, DNA:RNA, and RNA:DNA.
 3. The method of claim 2 wherein theanalyte:surface-bound ligand interaction pair is an antigen:antibodyinteraction pair.
 4. The method of claim 1 wherein the biosensor is anaffinity-based biosensor.
 5. The method of claim 4 wherein theaffinity-based biosensor is a surface plasmon resonance biosensor. 6.The method of claim 1 wherein the first liquid is an aqueous solutioncomprising at least one acidic, basic, ionic, organic, detergent orchelating agent.
 7. The method of claim 1 wherein the particulate solidcapturing material is separation beads.
 8. The method of claim 7 whereinthe separation beads are made from agarose, dextran, hydroxyapatit,silica, polyacrylamid, or hydrophilic polymers.
 9. The method of claim 8wherein the separation beads comprise chromatographic media havingspherical shapes with diameters ranging from 2 to 10 micrometers. 10.The method of claim 7 wherein the separation beads have magneticproperties.
 11. The method of claim 1 wherein the analyte:surface-boundligand interaction pair is a plurality of analyte:surface-bound ligandinteraction pairs, the step of eluting the analyte from thesurface-bound ligand generates a plurality of free analytes, the step ofcapturing generates a plurality of captured analytes, the consolidated,particulate solid capturing material has a plurality of capturedanalytes associated therewith, and a plurality of analytes are freedfrom the particulate solid capturing material.
 12. The method of claim11 wherein the step of consolidating comprises passing the particulatesolid capturing material, having the plurality of captured analytesassociated therewith and carried within the first liquid, through aseparation device that prevents passage of the particulate solidcapturing materials having the plurality of captured analytes associatedtherewith, while allowing passage of the first liquid, and, thereby,consolidating the plurality of captured analytes.
 13. The method ofclaim 12 wherein the separation device comprises a column, and the firstliquid containing the plurality of captured analytes is passed throughthe column.
 14. The method of claim 11, wherein the step of freeing theplurality of analytes from the particulate solid capturing material isaccomplished by eluting the plurality of captured analytes from theparticulate solid capturing material by contacting the consolidated andcaptured plurality of analytes with a second liquid that dissociates theplurality of captured analytes from the particulate solid capturingmaterial to yield a second liquid comprising a plurality of freeanalytes.
 15. The method of claim 14 wherein the second liquid is anaqueous solution comprising at least one acidic, basic, ionic, organic,detergent or chelating agent.
 16. The method of claim 14 wherein elutingthe plurality of free analytes from the particulate solid capturingmaterial occurs in a column employed to consolidate the particulatesolid capturing material having the plurality of captured analytesassociated therewith.
 17. The method of claim 14 wherein the secondliquid comprising the plurality of free analytes is collected, and theplurality of free analytes are subjected to subsequent analysis.
 18. Themethod of claim 1 wherein the removal of the particulate solid capturingmaterial to the location remote from the biosensor surface isaccomplished by flowingly carrying the solid capturing material, whilewithin the first liquid, away from the biosensor surface, then furthertransporting the solid capturing material to the remote location. 19.The method of claim 1 further comprising the step of subjecting thefreed analyte to a subsequent analysis.